Senile diseases such as osteoporosis are becoming preponderant with an ever increasing number of the elderly people. Osteoporosis is a bone disease that causes reduced bone mineral density (BMD), disrupted bone microarchitecture, and alteration of the amounts various proteins in bone, leading to an increased risk of fracture. Osteoporosis is caused by an imbalance between bone resorption and bone formation and progresses when bone resorption outpaces bone formation. With osteoporosis, the calcified bone tissue density decreases, causing the expansion of marrow cavity. As the symptoms progress, bone fracture occurs easily even by a slight stressful impact.
Early studies for osteoporosis had focused mainly on calcium and phosphorus metabolic disorders, but failed to define the pathogenesis thereof. The existing therapeutic agents for treating osteoporosis are represented by bisphosphonate products (e.g., Alendronate and Etidronate), hormone products (e.g., raloxifene), vitamin D products, calcitonin products, and calcium products. However, bisphosphonate products have the problems of a low absorption rate, a complicated method of administration, and the tendency to induce esophagitis. Hormone products require life-time administration, which may cause side effects such as breast cancer, uterine cancer, cholelithiasis, and thrombosis. Vitamin D products are expensive but not much effective. Calcitonin products have also the problems of high costs and an uneasy method of administration. Calcium products have fewer side effects but are limited to prevention rather than treatment. Short-term administration of a drug is not much effective for the treatment of osteoporosis, requiring long-term administration of a drug. Therefore, a novel drug causing much reduced side effects and having enhanced medicinal effects is required for long-term administration.
Bone marrow-derived adult stem cells, particularly mesenchymal stem cells (MSCs), are of a pluripotent cell type that differentiates into several distinct cell lineages, such as osteocytes, chondrocytes, myocytes, and adipocytes. The differentiation of adult stem cells tends to reduce the age-related diseases, to prevent: the reduction in the regeneration of bone, cartilage, and muscle tissues; the decline of the immune function; and environment-caused diseases. Thus, a method for activating the differentiation of adult stem cells may be an efficacious therapy for diseases associated with metabolic diseases, bone diseases, and aging.
The differentiation of adult stem cells into a specific cell type is controlled by an important transcription factor, the expression of which is regulated by the interaction with the outer signal transduction. In particular, adipocyte differentiation is known to be regulated by transcription factor PPARγ (peroxisome proliferation-activated receptor γ). The transcription factor facilitates or inhibits the adipocyte differentiation by binding with different ligands, and an increase in the activity of the transcription factor is known to facilitate the adipocyte differentiation, leading to increased obesity [MacDougald et al., Annu. Rev. Biochem., 1995; 64:345-73; Adams et al., J. Clin. Invest., 1997; 100:3149-53; Fajas et al., Curr. Opin. Cell Biol., 1998; 10:165-73].
Recently, a transcription coactivator, protein TAZ (transcription coactivator with PDZ-binding motif), which regulates the transcription factor PPAR γ has been identified [Kanai et al., Embo. J., 2000; 19:6778-91]. The TAZ protein is cloned as a partner protein which binds to 14-3-3 proteins, and it is phosphorylated at serine 89, which interact with 14-3-3 proteins in the cytosol [Kanai et al., Embo. J., 2000; 19:6778-91; Park et al., J. Biol. Chem., 2004; 279:17384-90]. The TAZ protein contains WW domains, coiled-coil domains, and PDZ-binding motifs, which suggests the possibility of various binding with other proteins. In particular, the WW domains show strong binding affinity with peptide sequence PPXY, which suggests the possibility of the TAZ protein to bind with several proteins containing PPXY motifs. In 2003, it was found that the WW domains in protein TAZ bind with RUNX2 (runt-related transcription factor 2) which is a decisive regulatory factor to facilitate the osteoclast differentiation. It was reported that the expression regulatory activity of RUNX2 target gene is amplified and the expression of bone-specific gene increases, leading to an osteogenic facilitation, through the binding [Hong et al., Science, 2005; 309:1074-8]. Further, polymavirus T antigen, a protein binding to the WW domains in protein TAZ is known, but its precise function in cells is not clearly understood. Furthermore, PPARγ, one of transcription factor having PPXY motif, has been identified as a novel TAZ binding protein, and such binding inhibits the adipocyte differentiation by PPARγ [Hong et al., Science, 2005; 309:1074-8]. The mechanism of protein TAZ for the inhibition of adipocyte differentiation is explained by the fact that protein TAZ binds with PPARγ not only to inhibit the DNA binding activity of PPARγ but also to inhibit the gene transcription facilitation activity, thus inhibiting the expression of adipocyte-specific PPARγ target gene. The binding of protein TAZ with RUNX2 and PPARγ plays a significant important role in regulating the differentiation of MSCs. Specifically, it has been found in the differentiation of MSCs that osteoclasts differentiation is facilitated, while the adipocyte differentiation is regressed by the binding of protein TAZ with proteins RUNX2 and PPARγ, [Hong et al., Science, 2005; 309:1074-8; Deng et al., Front Biosci., 2008; 13:2001-21; Hong et al., Cell Cycle 2006; 5:176-179]. That is, the differentiation of protein TAZ determines the differentiation of MSCs.
TBX5 (T-box transcription factor 5) is known as another TAZ binding protein, and the binding thereof is understood to play an important role in the limb and cardiac formation [Murakami et al., Proc. Natl. Acad. Sci., USA., 2005; 102:18034-9]. Further, TBX5 possibly binds with protein PAX3 important in the early embryonic phase to regulate its function [Murakami et al., Biochemical & Biophysical Research Communications, 2006; 339:533-9]. In addition, protein TAZ shows its activity by binding with various PDZ domain-containing proteins through PDZ-binding motif. TTF-1 (thyroid transcription factor-1) is a gene that plays an active role in the lung development, and regulates the surfactant protein-C gene expression. Protein TAZ is considered to be a transcription coactivator that facilitates the surfactant protein-C gene expression by binding with TTF-1 [Park et al., J. Biol. Chem., 2004; 279:17384-90]. Further, protein TAZ is suggested to regulate the expression of TEF-1 regulatory genes in muscle tissues by binding with TEF-1 (transcription enhancer factor-1) [Mahoney et al., Biochem. J., 2005; 388:217-25].
Aside from the TAZ function of modulating mesenchymal stem cell differentiation, other functions thereof in the migration, invasion, and tumorigenesis of breast cancer cells MCF7 are known [Chan et al., Cancer Res., 2008; 68:2592-8]. The occurrence of polycystic kidney disease which forms multiple renal cysts has been reported in TAZ-deficient animal models, and various approaches for the functions of protein TAZ have been attempted [Makita et al., Am. J. Physiol. Renal. Physiol., 2008; 294:F542-53; Tian et al., Molecular & Cellular Biology, 2007; 27:6383-95]. Meanwhile, there have been some reports that FGF-2 causes the decrease of the TAZ protein in osteogenic differentiation [Deng Z L et al., Front Biosci., 2008; 13:2001-21; Eda et al., Biochemical & Biophysical Research Communications, 2008; 366:471-5], but few investigations on the regulatory mechanism of the TAZ protein have been conducted despite the importance of functions of the TAZ protein.
In case of the TAZ protein which acts as a transcription co-activator to regulate transcription factors with DNA binding activity, the migration thereof from cytosol to nucleus is prerequisite. Until now, it is known that the TAZ protein can migrate into the nucleus by serine dephosphorylation, and the inhibition of binding between proteins TAZ and 14-3-3 is suggested as a method for increasing the migration of the TAZ protein into the nucleus. Thus, compounds which facilitate the migration of TAZ into the nucleus may be considered to have an inhibitory effect on the adipocyte differentiation and facilitates the osteoblast differentiation.
The present inventors have endeavored to seek out a compound effective for preventing or treating osteoporosis, diabetes, or hyperlipidemia, which has led the finding that phenyltetrazole derivatives are effective in the treatment or prevention of osteoporosis by regulating the TAZ protein and also in the treatment of the obesity by inhibiting adipocyte differentiation.